Neurodegenerative diseases, including Alzheimer's, Huntington's and Parkinson's Diseases and the Transmissible Spongiform Encephalopathies (prion diseases), have been linked to an unusual disease mechanism, in which alternative conformations of cellularly encoded proteins arise and subsequently direct the misfolding of normal conformers of the same protein to a like state. The spontaneous appearance of these disorders has been shown to increase with age, suggesting that cellular mechanisms exist to prevent the pathogenic misfolding pathways from initiating. Although the mechanism of conformational self-replication for these proteins has been well-studied, our mechanistic understanding of the initiation of this state lags far behind. Understanding the pathway that soluble, functional proteins take during this initial misfolding event can provide new therapeutic targets for the prevention and treatment of these diseases. The long-term goal of our research is to understand how the cellular environment regulates protein folding pathways to create both normal physiological and disease states. The objective of this proposal is to determine how a pathway of protein misfolding is initiated in vivo. For these studies, we will exploit the Sup35/[PSI+] prion system of Saccharomyces cerevisiae. While the mechanism by which Sup35 folding switches from the normal ([psi-]) to the misfolded ([PSI+]) pathway is still unknown, this process is dependent on presence of the misfolded form of another prion protein, Rnq1, or on a Sup35 variant with a non-native C- terminal extension. We hypothesize that these factors bypass the rate-limiting step for accumulation of misfolded Sup35, leading to [PSI+] appearance. To test this hypothesis, we will manipulate both the expressing levels of key players in the Sup35 misfolding pathway and the Sup35 sequence and link changes in the frequency of [PSI+] appearance to changes in the biogenesis of the Sup35 protein in vivo. By developing an understanding of how protein folding pathways can be modulated by their cellular environment, we will begin to reveal the molecular contributions of these processes to both normal cellular physiology and to the appearance and spread of diseases.